RAS CAAX-motif modifying proteins

The C-terminal CAAX motif of Ras, and numerous small GTPases undergoes post-translational modification by a sequence of enzymatic reactions that determines the ultimate localisation of Ras to the cell membrane.

Interfering with these processes causes mis-localisation of Ras and blocks its ability to mediate growth factor-mediated signal transduction processes and the capacity of oncogenic Ras to transform cells. We have focused our efforts on obtaining structural and mechanistic insights into two enzymes involved in this process, RCE1 and ICMT, two intrinsic membrane proteins localised to the endoplasmic reticulum.

RCE1 (RAS concerting enzyme 1)

We undertook high throughput expression screening of ~40 RCE1 homologs from eubacteria and archaea (including thermophilic prokaryotes) to identify proteins suitable for crystallographic analysis using GFP screening approaches.

We identified an RCE1 from a bacterial organism with ~25% sequence identity to the human counterpart (including conservation of predicted catalytic residues). RCE1 has been crystallised, and recently well-ordered diffracting crystals have been obtained which diffract to ~7 Å resolution.

ICMT (isoprenyl cysteine methyltransferase)

Similarly to RCE1 we obtained ~40 prokaryotic ICMT homologs for expression screening using GFP as an assay of folding and expression levels. We obtained promising expression with 3 or 4 homologs, one of which can be expressed, purified and crystallised. Single crystals diffract to ~6 Å resolution and optimisation and structure determination is in progress.